2.4 | Ultrasound-induced cytosolic delivery of β-Gal
To demonstrate ultrasound-induced cytosolic delivery of proteins, β-Gal was carried into DLD1 cells by conjugation with 9E5-conjugated PCNDs. After the intracellular accumulation of PCNDs carrying β-Gal, the cells were exposed to ultrasound using previously reported experimental setup[30]. The cytosolic release of β-Gal was visualized using a fluorogenic substrate, namely TokyoGreen-βGal (TG-βGal)[34]. In addition, to visualize endosomes and lysosomes in the endocytosis pathway, the cells were stained with Lysotracker® Blue, which helps in visualizing acidic organelles. In ultrasound-exposed cells, green fluorescence derived from the hydrolysis of TG-βGal was observed throughout the intracellular area (Figure 3A), as previously reported in β-Gal-expressing cells[34]. This result clearly showed that β-Gal was released into the cytosol. In contrast, in non-ultrasound-exposed cells, fluorescence was observed only in endosomes and lysosomes (Figure 3B). Similarly, cytosolic fluorescence was not observed in the control cells, which were treated with PCNDs without β-Gal after the ultrasound treatment (Figure 3C); the fluorescence was also not observed in the untreated cells (Figure 3D). This result clearly indicated that β-Gal was released from the endocytosis pathway into the cytosol in response to ultrasound treatment.